This invention relates generally to the bioremediation of contaminated soil, and ground water but may be applied to any application in which a media is intended to be transformed by the bioremediation processes and capabilities resulting from the growth and metabolism of Fungi and or Monera, and the liberation of their associated enzymes and chemical exudates. The invention is more particularly a method for the growing Fungi or Monera in situ where by the device is consumed by Fungi or Monera or a consortium of organisms, and produces enzymes that digest the target media such as crude oil in soil or contaminants in associated ground water.
Wood and Bamboo are particularly good materials for constructing the device as they are rigid, easily formed into hollow tubes that can be driven into contaminated soil and are easily inoculated with desirable species of Fungi or Monera that are capable of digesting a wide range of contaminants or target substrates.
Naturally-occurring and intentionally designed Fungi and Monera colonize and digest wood and other organic fibers. They accomplish this digestion and degradation process by exuding enzymes and colonizing the cell matrix, composed of cellulose, hemicellulose, lignin and other carbonaceous and protein compounds. These enzymes and chemical exudates are also capable of digesting almost any organic compound and rendering it into carbon dioxide, water, nitrogen gas and minerals.
These Fungi and Monera have been found to be effective in sequestering heavy metals from contaminated soil or water and in reducing certain metalloids to their elemental form. The art of mycoremediation and mycorestoration has been refined by a number of practitioners.
Mycoremediation is a form of bioremediation, the process of using mushrooms to return an environment (usually soil) contaminated by pollutants to a less contaminated state. The term mycoremediation was coined by Paul Stamets and refers specifically to the use of fungal mycelia in bioremediation. Mycelium is the vegetative part of a fungus, consisting of a mass of branching, thread-like hyphae. The mass of hyphae is sometimes called shiro, especially within the fairy ring fungi. Fungal colonies composed of mycelia are found in soil and on or in many other substrates. Mycelium may form fruiting bodies such as mushrooms. A mycelium may be minute, forming a colony that is too small to see, or it may be extensive.
It is through the mycelium that a fungus absorbs nutrients from its environment. It does this in a two stage process. Firstly the hyphae secrete enzymes onto the food source, which breaks down polymers into monomers. These monomers are then absorbed into the mycelium by facilitated diffusion and active transport. A hypha (plural hyphae) is a long, branching filamentous cell of a fungus, and also of unrelated Actinobacteria. In fungi, hyphae are the main mode of vegetative growth, and are collectively called a mycelium.
A hypha consists of one or more cells surrounded by a tubular cell wall. In most fungi, hyphae are divided into cells by internal cross-walls called septa (singular septum). Septa are usually perforated by pores large enough for ribosomes, mitochondria and sometimes nuclei to flow among cells. The structural polymer in fungal cell walls is typically chitin (in contrast plants have cellulosic cell walls, and animal cells lack walls). Some Fungi however, have non septate hypha, meaning their hypha are not separated by septa.
Hyphae grow at their tips. During tip growth, cell walls are extended by the external assembly and polymerization of cell wall components, and the internal production of new cell membrane. The Spitzenkorper is an intracellular organelle associated with tip growth. It is composed of an aggregation of membrane-bound vesicles containing cell wall components. The vesicles travel to the cell membrane via the cytoskeleton, and dump their contents outside the cell by the process of exocytosis. Vesicle membranes contribute to growth of the cell membrane while their contents form new cell wall. As a hypha extends, septa may be formed behind the growing tip to partition each hypha into individual cells. Hyphae can branch through bifurcation of a growing tip, or by the emergence of a new tip from an established hypha.
Hyphae may be modified in many different ways to serve specific functions. Some parasitic fungi form haustoria that function in absorption within the host cells. The arbuscules of mutualistic mycorrhizal fungi serve a similar function in nutrient exchange, so are important in assisting nutrient and water absorption by plants. Hyphae are found enveloping the gonidia in lichens, making up a large part of their structure. In nematode-trapping fungi, hyphae may be modified into trapping structures such as constricting rings and adhesive nets. Cords can be formed to transfer nutrients over larger distances.
Mycelium is vital in terrestrial and aquatic ecosystems for its role in the decomposition of plant material. It contributes to the organic fraction of soil and its growth releases carbon dioxide back into the atmosphere. The mycelium of mycorrhizal fungi increases the efficiency of water and nutrient absorption of most plants and confers resistance to some plant pathogens. Mycelium is an important food source for many soil invertebrates. Sclerotia are compact or hard masses of mycelium.
One of the primary roles of fungi in the ecosystem is decomposition, which is performed by the mycelium. The mycelium secretes extracellular enzymes and acids that break down lignin and cellulose, the two main building blocks of plant fiber. These are organic compounds composed of long chains of carbon and hydrogen, structurally similar to many organic pollutants. The key to mycoremediation is determining the right fungal species to target a specific pollutant. Certain strains have been reported to successfully degrade the nerve gases VX and sarin. Enzymes are proteins that catalyze (i.e. accelerate) chemical reactions. In enzymatic reactions, the molecules at the beginning of the process are called substrates, and the enzyme converts them into different molecules, the products. Almost all processes in a biological cell need enzymes in order to occur at significant rates. Since enzymes are extremely selective for their substrates and speed up only a few reactions from among many possibilities, the set of enzymes made in a cell determines which metabolic pathways occur in that cell.
Like all catalysts, enzymes work by lowering the activation energy (Ea or ΔG555) for a reaction, thus dramatically accelerating the rate of the reaction. Most enzyme reaction rates are millions of times faster than those of comparable uncatalyzed reactions. As with all catalysts, enzymes are not consumed by the reactions they catalyze, nor do they alter the equilibrium of these reactions. However, enzymes do differ from most other catalysts by being much more specific. Enzymes are known to catalyze about 4,000 biochemical reactions. Although almost all enzymes are proteins, not all biochemical catalysts are enzymes, since some RNA molecules called ribozymes also catalyze reactions. Synthetic molecules called artificial enzymes also display enzyme-like catalysis.
Enzyme activity can be affected by other molecules. Inhibitors are molecules that decrease enzyme activity; activators are molecules that increase activity. Many drugs and poisons are enzyme inhibitors. Activity is also affected by temperature, chemical environment (e.g. pH), and the concentration of substrate. Some enzymes are used commercially, for example, in the synthesis of antibiotics. In addition, some household products use enzymes to speed up biochemical reactions (e.g., enzymes in biological washing powders break down protein or fat stains on clothes; enzymes in meat tenderizers break down proteins, making the meat easier to chew).
In an experiment conducted in conjunction with Battelle, a major contributor in the bioremediation industry, a plot of soil contaminated with diesel oil was inoculated with mycelia of oyster mushrooms; The Oyster mushroom, or Pleurotus ostreatus, is a common mushroom prized for its edibility. Long cultivated in Asia, it is now cultivated around the world for food. It is related to the similarly cultivated “king oyster mushroom”. Oyster mushrooms can also be used industrially for mycoremediation purposes. Traditional bioremediation techniques (bacteria) were used on control plots. After four weeks, more than 95% of many of the PAH (polycyclic aromatic hydrocarbons) had been reduced to non-toxic components in the mycelial-inoculated plots. It appears that the natural microbial community participates with the fungi to break down contaminants, eventually into carbon dioxide and water. Wood-degrading fungi are particularly effective in breaking down aromatic pollutants (toxic components of petroleum), as well as chlorinated compounds (certain persistent pesticides; Battelle, 2000).
Saprophytic and parasitic fungi help create the organic components of topsoil, with the interdependent assistance of bacteria, insects, and other organisms. An array of primary, secondary, and tertiary saprophytic fungi convert wood and plant materials into biodynamic soil components. These soils benefit plants that in turn use photosynthesis to manufacture their own foods. They are capable of changing the pH and weathering the soil and parent rock underlying it.
Work performed by the Institute of Microbiology, Academy of Sciences of the Czech Republic, 142 20 Prague, Czechia. Identified a number of white-rot fungal cultures, strains of Irpex lacteus and Pleurotus ostreatus which were selected for degradation of 7 three- and four-ring unsubstituted aromatic hydrocarbons (PAH) in two contaminated industrial soils. Respective data for removal of PAH in the two industrial soils by I. lacteus were: fluorene (41 and 67%), phenanthrene (20 and 56%), anthracene (29 and 49%), fluoranthene (29 and 57%), pyrene (24 and 42%), chrysene (16 and 32%) and benzo[a]anthracene (13 and 20%). In the same two industrial soils P. ostreatus degraded the PAH with respective removal figures of fluorene (26 and 35%), phenanthrene (0 and 20%), anthracene (19 and 53%), fluoranthene (29 and 31%), pyrene (22 and 42%), chrysene (0 and 42%) and benzo[a]anthracene (0 and 13%). The degradation of PAH was determined against concentration of PAH in non-treated contaminated soils after 14 weeks of incubation. The fungal degradation of PAH in soil was studied simultaneously with ecotoxicity evaluation of fungal treated and non-treated contaminated soils. Compared to non-treated contaminated soil, fungus-treated soil samples indicated decrease in inhibition of bioluminescence in luminescent bacteria (Vibrio fischerii) and increase in germinated mustard (Brassica alba) seeds.
Mycofiltration is a similar or same process, using fungal mycelia to filter toxic waste and microorganisms from water in soil.